Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM

Masanori Osawa, Alexandra Dace, Kit I. Tong, Aswani Valiveti, Mitsuhiko Ikura, James B. Ames

研究成果: Article査読

94 被引用数 (Scopus)

抄録

DREAM (calsenilin/KChlP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca2+ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca2+ binding to the various mutants revealed that, in the absence of Mg2+, Ca2+ binds independently and se-quentially to EF-3 (ΔH = -2.4 kcal/mol), EF-4 (ΔH = +5.2 kcal/mol), and EF-2 (ΔH = +1 kcal/mol). By contrast, only two Ca2+ bind to DREAM in the presence of physiologi-cal levels of Mg2+ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg2+. ITC measurements demonstrate that one Mg2+ binds enthalpically with high affinity (Kd = 13 μM and ΔH = -0.79 kcal/mol) and two or more Mg2+ bind entropically in the millimolar range. Size-exclusion chromatograpliy studies revealed that Mg2+ stabilizes DREAM as a monomer, whereas Ca2+ induces protein dinierization. Electrophoretic mobility shift assays indicated that Mg2+ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca2+ levels, suggesting that binding of a single Ca 2+ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca2+ and Mg2+ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg2+ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca 2+-induced protein dimerization disrupts DNA binding.

本文言語English
ページ(範囲)18008-18014
ページ数7
ジャーナルJournal of Biological Chemistry
280
18
DOI
出版ステータスPublished - 2005 5 6
外部発表はい

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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